Properties of the Bimodal Fluorescent Protein Produced by Photobacterium phosphoreum

Abstract

A fluorescent protein isolated from the deep‐sea luminous bacterium Photobacterium phosphoreum strain bmFP has been purified, cloned and sequenced. The protein is 96.5% identical in amino acid sequence to FP390, the weakly fluorescent flavoprotein encoded by the luxF gene characteristic of Photobacterium species. Similar to FP390, bmFP is a dimer of two homologous subunits binding four FMN‐myristate chromophores but has the distinctive feature of emitting a bimodal fluorescence with maxima at about 488 and 517 nm, hence the name bmFP. For both bands of this fluorescence, the excitation spectrum exhibits a peak at 336 nm, not corresponding to its flavin‐like absorption spectrum. Heating of bmFP in urea resulted in a decrease in the intensity of the 488 nm band along with the appearance of a new fluorescence peaking at 423 nm, partially reversible upon the removal of the urea. Upon complete denaturation, either by heat or guanidium chloride at 65°C, fluorescence characteristic of both free flavin and this 423 nm species appears. It is speculated that chromophores in different states of protonation, associated with a single protein, are responsible for the unusual spectral properties of bmFP.

Phylogenetic Diversity and Cosymbiosis in the Bioluminescent Symbioses of “Photobacterium mandapamensis”

“…The mutations provide a natural test of the functional role of LuxF in luminescence. LuxF, a nonfluorescent flavoprotein (FP 390 ), exhibits substantial amino acid sequence similarity to the ␣ and ␤ subunits of luciferase, especially to the ␤ subunit, and it is coexpressed with LuxA, LuxB, and other lux operon proteins, suggesting that LuxF plays a role in light emission (20,23,30,37). We found that strains carrying nonsense mutations in luxF are luminous, but less strongly so than other strains, and that the lower production of light probably is not due to a limitation for aldehyde, a substrate for the luminescence reaction (see the supplemental material).…”